In Vitro Apoptotic Effects of Farnesyltransferase blockade in Acute Myeloid Leukemia Cells.

Farnesyltransferase inhibitors (FTIs) are a class of oral anti-cancer drugs currently tested in phase I-II clinical trials for treatment of hematological malignancies. The in vitro effects of various FTIs (alpha-hydroxyfarnesylphosphonic acid, manumycin-A and SCH66336) were tested on CD34+ KG1a cell line and in primary acute myeloid leukemia (AML) cells from 64 patients. By cell viability and clonogeneic methylcellulose assays, FTIs showed a significant inhibitory activity in CD34+ KG1a and primary bone marrow (BM) leukemic cells from 56% of AML patients. FTIs also induced activation of caspase-3 and Fas-independent apoptosis, confirmed by the finding that inhibition of caspase-8 was not associated with the rescue of FTI-treated cells. We concluded that other cellular events induced by FTIs may trigger activation of caspase-3 and subsequent apoptosis, but the expression of proapoptotic molecules, as Bcl-2 and Bcl-XL, and antiapoptotic, as Bcl-X(s), were not modified by FTIs. By contrast, expression of inducible nitric oxide synthase (iNOS) was increased in FTI-treated AML cells. Our results suggest a very complex mechanism of action of FTIs that require more studies for a better clinical use of the drugs alone or in combination in the treatment of hematological malignancies.


In Vitro Apoptotic Effects of Farnesyltransferase blockade
in Acute Myeloid Leukemia Cells Università degli Studi di Salerno may be not necessary for FTI antineoplastic effects [40][41].
The in vitro cytotoxic effects of various FTIs have been previously described, including the NOmediated apoptotic effects of SCH66336 in CML cells [42][43], but FTIs alone have shown only limited activity in AML and MDS patients [44][45][46]. The aim of the present study was to clarify the mechanism of action of FTIs, in order to explain the not enthusiastic results obtained in in vivo experiments. Consequently, we studied the in vitro effects of FTIs on apoptosis and growth of AML primary cells by blocking the downstream and upstream proteins of Ras-mediated apoptosis pathways.

Suspension cultures
KG1a and primary AML cells were placed in 24well plates in RPMI containing 0.1% FCS for 12 and 2 hours, respectively, before exposure to FTIs. For functional experiments, AML cells were preincubated for 2 hours with the following reagents: caspase-3 inhibitor Z-DEVD-FMK, caspase-8 inhibitor IETD-FMK, (all used at 50 µM and purchased from Alexis, San Diego, CA), Fasreceptor triggering inhibitor Fas:Fc (50 µM) (Alexis), iNOS inhibitor NG-monomethyl-arginine (500 µM) (γ-MM-arg; Calbiochem, San Diego, CA). All experiments were repeated at least 3 times and each experimental condition was repeated at least in duplicate wells in each experiment. All the incubations were conducted at 37°C with 5% CO2.

Proliferation assay
In vitro sensitivity of KG1a and primary BM AML cells to FTIs was determined by plating 5 x 10 5 cells in RPMI 0.1% FCS and several dilutions of FTIs in 24well plates. Controls were performed using identical concentrations of the solvent used for FTIs. After 48h incubation, cell viability was determined by MTS (3-(4,5dimethylthiazole-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and PES (phenazine ethosulfate) assay (CellTiter 96 AQueous One Solution Reagent) provided by Promega (Madison, WI, USA). Briefly, 20 l of CellTiter 96 AQueous One Solution Reagent was added to each well and incubated for 4 hours. The optical density (OD) was measured at 490 nm using a microplate spectrophotometer (Titertek Multiscan, BioRad, Hercules, CA). FTI effects were measured as percentage of inhibition of cell viability by the following equation: 1-[(OD treated well/mean OD control wells) x 100], after background correction using blank wells. Each experiment was performed in triplicate. Values were expressed as mean value ± standard error of mean (SEM). The results were considered evaluable only if the control wells contained at least 70% of live AML cells after 48 h culture.

Hematopoietic colony assay
Short-term clonogenic progenitors were measured in methylcellulose (Stem Cell Technologies, Vancouver, CA). Isolated BMMNCs were plated in methylcellulose at 1 x 10 3 cells/ml concentration (35 mm dishes; 1 ml of medium/dish) in the presence of the following cocktail: 10 ng/mL IL-3, 50 ng/mL G-CSF, 50 ng/mL GM-CSF, 20 ng/mL SCF, and 2 U/mL EPO. Anti-Fas antibody (clone CH11, Amac) was used at concentrations ranging from 20-1000 U/ml to 1 µg/ml. Myeloid and erythroid colonies and clonogeneic aggregates of less than 50 cells, were counted as granulocyte-macrophage colony forming unit (CFU-GM), erythroid-burst forming unit (BFU-E) and clusters, respectively, after 14-day incubation. Each experiment was done in quadruplicate. Values were expressed as mean value ± standard error of mean (SEM).

Apoptosis assays
DNA fragmentation was measured after low molecular weight (LMW) DNA extraction from 2 x 10 6 cells as previously described [48]. The high molecular weight DNA fraction was precipitated for 6 h in the presence of 5 mol/L NaCl and pelletted by high-speed centrifugation. The fragmented DNA was extracted, precipitated and electrophoresed as previously described [48]. Università degli Studi di Salerno Viability and cluster/colonies of AML cells were detected by MTT (A) and methylcellulose assays (B) measured by colony forming cell (CFC) assays. Each dot (subject studied) represents the percentage of inhibition of cell viability and colony formation by each FTI at the IC50 described in the text (control cultures were considered 100%). Horizontal bars are the mean values, and vertical bars are SEMs. Cumulative mean inhibition of cell viability and CFC ± SEM, as well as statistical analysis, are reported in the corresponding results section.
Abbreviations, see text.
Flow cytometric analysis of cellular DNA was performed using propidium iodide (PI) staining as previously described [48], adding 100 U/mL RNAse A (Boehringer Mannheim, Mannheim, Germany) during the incubation step. A minimum of 60,000 events was counted per sample. Apoptotic cell nuclei containing hypodiploid (fragmented) DNA were counted as a percentage of total population.

Detection of caspases 3 and 8 activities
Activation of intracellular caspases was performed by flow cytometry using fluorogenic caspasespecific substrates (DEVD-AFC for caspase-3, Alexis; and IETD-AFC for caspase-8, Pharmingen). Cells (1 x 10 6 ) were treated with FTIs in the presence or absence of caspase-specific inhibitors for 24 hours, washed with PBS, and then resuspended in 50 µL of substrate buffer containing 10 mmol/L dithiothreitol (DTT) and 10 µL of the fluorogenic caspase-specific substrate supplemented with 5 µL of FCS. After centrifugation at 800g for 5 minutes, cells were incubated at 37°C for 60 minutes. After incubation, cells were washed and acquired. At least 10,000 events were analysed. Results were measured as fold increase in fluorescence relative to untreated control cells.

Immunoblotting of iNOS, Bcl2, BclXL and p53
Treated AML cells were washed and lysed as previously described [48]. Concentrations were measured by colorimetric method (Biorad, Richmond, CO). A total of 100 µg of cell lysate, together with molecular weight markers (Amersham, Little Chalfont, UK) and iNOS positive mouse macrophages lysate (Transduction Laboratories, Lexington, KY) were fractionated by 7.5% SDS-polyacrylamide gel electrophoresis. PVDF membranes were prepared as previously described [48] and incubated with 2 µg/ml of mouse anti-iNOS, anti-Bcl2, anti-p53, anti-BclXL and anti-BclXS antibodies (Transduction Laboratories, Lexington, KY) overnight at 4°C. The reaction was revealed by incubating filters with horseradish peroxidase-conjugated goat anti-rabbit antibodies (BioRad, Richmond, CO) and developed by ECL (Amersham, Little Chalfont, UK) according to manufacturer's specifications.

Statistical analysis
Two-tailed Student t-test was used for flow cytometric analysis and tissue culture experiments. p values  0.05 were considered statistically significant.

FTI-induced growth inhibition of AML cells is partly related to apoptosis
We first investigated the effects of FTIs on growth of KG1a AML cell line. In cell viability tests performed in logarithmic growth phase, KG1a cell survival was inhibited by all three FTIs in a dose Università degli Studi di Salerno dependent manner. By linear regression analysis, we determined the 50% inhibitory concentration (IC50) after 48 hour exposure to SCH, Man-A and α-HFPA. On an equimolar concentrations, SCH displayed more toxicity than Man-A (IC50 mean, 5 µM vs 50 µM, respectively; range, 1-20 µM vs 25-150 µM, respectively) and α-HFPA (IC50 mean, 100 µM; range, 75-150). The observed mean IC50 concentrations were used for next experiments.
The inhibitory effects of SCH, Man-A and α-HFPA were significantly stronger in primary AML cells than normal marrow cells (mean inhibition % ± SEM, 35±6 vs 13±5, respectively; p=0.03). We documented a FTI-mediated inhibition of cell viability >25% in 56% of AML patients (Fig. 1A). Similarly, by methylcellulose clonogeneic assays, colony and cluster growth inhibition >25% was observed in 64% of AML patients, whereas colony cells from normal BM cells were only weakly affected by FTIs exposure (mean inhibition % ± SEM, 47.7±7 vs 15.5±1, respectively; p<0.001) (Fig. 1B). By allele-specific PCR, the presence of oncogenic N-Ras and K-Ras mutations were also investigated in primary cells from 30 AML patients. Oncogenic mutations of N-Ras were detected in 27% of AML subjects; by contrast, we did not observe K-Ras mutations in any AML cell sample. Indeed, FTI-mediated inhibition of cell growth was observed in both AML with and without N-Ras mutations (data not shown).
To define whether the FTI-mediated growth inhibition of primary AML cells was associated to apoptosis, BM AML cells sensitive to FTI inhibition were exposed for 48 h to FTIs at IC50. By LMW DNA fragmentation analysis, AML cells showed the characteristic DNA ladder suggestive of apoptosis ( Fig.  2A). Flow cytometric detection of apoptotic hypodiploid DNA peak derived from treated BM AML cells confirmed the FTI-enhanced apoptotic cytotoxic effect (Fig. 2B). Indeed, percentages of apoptotic AML cells were significantly lower than those with cytolysis measured by cell viability assay (mean % ± SEM of cytolysis, 55.7±10; p=0.03). The parallel measurement of the number of apoptotic AML cells and viable cells from 10 AML patients carried by flow cytometry and cell viability assays showed that the percentages of apoptotic AML cells were significantly lower than those undergoing cytolysis (mean % ± SEM of apoptotic and viable AML cells, 32 ± 7 vs 51 ± 11, respectively; p = 0.03) (Fig. 2C).

Caspase-3 and Fas-independent pathways are involved in FTI-mediated apoptosis of AML cells
FTIs could induce caspase-3 activation by flow cytometric measurement of MFI after cleavage of the specific fluorogenic substrate DEVD-AFC (mean percentage of positive cells after 6 and 24h culture, 25±4% and 38±4% vs 9±1% and 18±5%, FTI-treated and control AML cells, respectively; mean of 5 experiments). In addition, pre-incubation with caspase-3 inhibitor Z-DEVD-FMK allowed to partially abrogate FTI-mediated caspase activation (mean % ± SEM after 24 h exposure to

Figure 2. FTI-induced inhibition of AML cell proliferation is partly related to apoptosis.
Agarose gel stained with ethidium bromide after electrophoresis of low molecular weight DNA from 12 representative BM AML patients exposed for 48 h to FTIs at the IC50 (A). A representative flow cytometric detection of apoptic hypodiploid DNA peak stained with PI from BM AML patient exposed to SCH (B). Percentages of viable cells and apoptotic cells simultaneously measured from 10 representative BM AML patients after exposure for 48 h to FTIs (C). Cumulative percentage of mean inhibition of cell viability and apoptotic cells, as well as statistical analysis, are reported in the corresponding results section.

FTIs trigger iNOS expression in AML cells
Unstimulated primary total AML BM cells expressed iNOS mRNA by PCR detection. Because iNOS expression in total BM AML cells might be related to non-leukemic cells, immature CD34 + KG1a cells were tested for the expression of iNOS mRNA after 48 h culture. A stronger amplification of iNOS was detected, and also after FTIs stimulation (Fig. 5A). Similarly, immunoblot of cell lysates showed lower levels of iNOS at baseline, and a 10-fold increased after FTIs stimulation in both primary total BM AML cells and KG1a cells (Fig.  5B). Using the cell-permeable fluorescent indicator DAF-2 DA, NO levels increased 40% after FTI exposure from basal levels detected in AML BM cells cultured in control medium without FTI (p<0.001), and γ -MM-arg partially blocked FTI-mediated NO production in CML cells ( Fig  5C). Inhibition of NO synthesis by pretreatment of AML cells with γ-MM-arg (500 µM), a competitive inhibitor of iNOS, did not affect the inhibitory effect on cell growth and apoptosis of FTIs (data not shown). However, when FTIs were used at IC10, γ-MM-arg partially prevented FTI effects (mean % ± SEM cell growth and apoptosis after FTI exposure, 31±2 and 21±4 vs 46±1 and 31±3 in absence and in presence of γ-MM-arg, respectively; p=0.01 and p=0.03) (Fig. 5C).

FTI exposure may modify p53 but not Bcl-2 pathway in AML cells
To explore whether FTI-induced apoptosis could be mediated by decreased expression of anti-apoptotic Bcl-2 and Bcl-X(L) or increasing in proapoptotic Bcl-X(S) proteins, AML cells were exposed for 48 hours to FTIs or control medium and Bcl-2 or Bcl-X(L)/(S) were measured. Quantification of protein banding by densitometry did not document changes in Bcl-2 protein expression in KG1a as well as in four primary BM AML samples after FTI exposure as compared to control cultures (Fig. 6A). Similarly, the expression of Bcl-X(L) and Bcl-X(S) did not show variations after FTI exposure Actin is used as control (B). γ-MM-arg partially preventes FTI-mediated apoptosis. Flow cytometric detection of apoptic hypodiploid DNA peak stained with PI from a CML patient treated with FTI (C). Università degli Studi di Salerno (Fig. 5B). By contrast, in KG1a cells and few AML cases, an enhancement of p53 expression after FTI exposure was detected (Fig. 6C).
Here, the in vitro effects of FTIs were studied in AML cells. We have shown that FTIs showed a significant inhibitory activity on cell viability in CD34 + KG1a cell line and primary BM cells from 56% of AML patients. Indeed, only half of our AML patients showed increased sensitivity of cluster and colony growth after FTI exposure compared to normal marrow progenitor cells. Furthermore, the lack of response in the remaining half of AML patients could be related to the presence of mutations in FTase or other markers of resistance to FTIs, as p53 mutations, but more studies are needed to confirm this hypothesis [49][50][51].
Preliminary data have excluded that FTImediated inhibition of cell growth was Ras-mutations dependent, as FTIs induced inhibition of viability in AML cells with and without N-Ras mutations (Selleri et al., personal communication). As previously reported for CML cells, FTI-mediated cytotoxic effects in AML cells were partially related to enhanced apoptosis [48]. Although it has been reported that Ras-transformed cells showed Fas-R upregulation and higher Fas-related apoptosis after FTI-exposure [52][53], we documented a Fas-independent FTI-mediated apoptosis in AML cells. Moreover, FTI-mediated apoptosis seemed to be caspase-3 dependent but caspase-8 independent as inhibition of caspase-8 was not associated with the rescue of FTItreated cells. These findings suggest that other cellular events induced by FTIs may trigger caspase-3 activation and subsequent apoptosis in AML cells. As the main event for caspase-3 activation is the release of cytochrome c from mitochondria, the expression of potential molecules modulating apoptosis via mitochondrial pathways were studied [54][55]. Expression of proapoptotic Bcl-2 and Bcl-X(L) and antiapoptotic Bcl-X(S) proteins were not modified by FTIs, except for the involvement of Bcl-2 pathway in FTI-induced apoptosis in human AML cells.
Another known mechanism that can concur in the inhibition of tumor growth is the macrophage-mediated NO release in the site of inflammation or in tumor environment, causing mitochondrial release of cytochrome c and apoptosis. In addition, it has been described that Ras inhibitors can increase NO-induced cell death [56][57][58][59][60][61][62]. In a similar manner, FTIs can induce apoptosis in CML cells, through cytochrome c release and caspase-3 activation, as previously documented [59,62]. As expected, FTIs also can induce iNOS expression in AML cells. Moreover, inhibition of NO synthesis partially abrogated the effects of FTIs on apoptosis suggesting that iNOS cascade may be only one of the possible mechanisms of FTI-mediated apoptosis in AML cells (Fig. 7 C-D). The enhancement of p53 expression in KG1a cell line and some AML cases suggested a complex mechanism of action of FTIs. Indeed, Moasser et al. has already reported a more susceptibility to FTIs in p53 wildtype breast cancer cell lines [63]. Because caspases, iNOS and p53 play an important role in several intracellular pathways and can be triggered by several different extracellular signals, it is hard to clearly understand the FTIs mechanisms, also due to their aspecific action [54][55][56][57][58][59][60][61][62]. Several pathways are deregulated in cancers, as JAK/STATs or Syk/Btk, allowing proliferation and survival of tumor cells [64][65][66][67]. Indeed, it has been reported that over-activity of growth factor signaling pathways in breast cancer did not correlate with sensitivity to FTIs [63]. On the other hand, FTIs have shown contradictory results in MDS an AML patients, particularly in elderly and poor-risk AML [68][69][70][71][72], but synergized actions were reported when FTIs were combined to tyrosine kinase inhibitors (TKIs) or other drugs, as simvastatin [73][74][75][76][77]. These findings suggest that FTIs can induce growth inhibition not only through apoptosis, but also interfering with proliferation pathways, enhanced when TKIs are used. Besides, the efficacy of ruxolitinib, a JAK1/2 inhibitor, or ibrutinib, a Btk inhibitor, has been described in several hematological malignancies, and more studies are ongoing [78][79]. For these reasons, to improve the therapeutic potential of FTIs alone or in combination with standard chemotherapy or new targeting therapies, it is important to better understand their mechanisms of action, as to investigate how they could impact on other pathways or vice versa.

V. CONCLUSION
As already reported, in vitro FTIs effects on cell viability and apoptosis are caspase-3 and iNOS dependent in AML cells, and Fas-R/Fas-L or Bcl2 independent. But the involvement of p53 suggests a more complex mechanism in the susceptibility of FTI-mediated apoptosis in AML cells. It is also possible that FTIs may have only additive anti-neoplastic effects, requiring the combination with other anti-cancer agents to be effective in AML therapy. Our data open new questions about FTIs mechanism of action and their use in myeloid malignancies as single agents or in combination, in order to improve the outcome of hematological patients who cannot be treated with more aggressive treatments.